This application claims priority to Indian Application No. 3504/DEL/97, filed Dec. 8, 1997.
1. Field of the Invention
This invention relates to a process for the preparation of reduced calorie fats. This invention particularly relates to the preparation of reduced calorie fats by incorporating behenic acid into edible oils such as sunflower, groundnut, safflower, rapeseed, soybean and fish oils. More particularly, it provides a reduced calorie plastic for containing essential fatty acids and natural antioxidants using different processes involving chemical interesterification of 1,3-dibehenin and edible oils or chemical interesterification of 1,3-dibehenin based structured fat and edible oils or enzymic transesterification of edible oils with alkyl behenates.
2. Background
Typical fats and oils provide approximately 6 kcal/g of metabolizable energy compared to 4 kcal/g for protein or carbohydrate [Atwater, W. O. et al., Annu. Rep. Storrs Agric. Exp. Stn. (1903) No.15, 123-146 and Maynard, L. A., J. Nutr. 28 (1944) 443-453]. In addition to the caloric and nutritional value, fats have many functions in the diet. Fats and oils carry, enhance and release the flavours of other food components, delays digestion, increases palatability of food and imparts the feeling of satiety. Certain unsaturated fatty acids like 9,12-octadecenoic acid (linole acid) which are known to be essential fatty acids are necessary as they are not produced in the body. Fats and oils are also associated with the fat-soluble vitamins A, D, E and K, and the absorption of these vitamins is impaired at very low fat intakes. Fat is also associated with diseases such as coronary heart disease and cancer, a high fat diet being positively linked to both. The U.S. Surgeon General has recommended that no more then 30% of the dietary calories should be derived from fat [U.S. Department of Health and Human Services, The Surgeon General""s Report on Nutrition and Health, DHHS (PHS) Publication 88-50210, [U.S.GPO., Washington, D.C. (1988)]. Regulatory and advisory bodies advocate a lowered fat intake in order to reduce the incidence and morbidity of many coronary diseases, stroke, high blood pressure, obesity and diabetes.
However, food habits are difficult to change and the positive contribution of fats to increase the palatability of foods is generally recognized. The level of fat in the diet of affluent societies is too high and needs to be lowered. Reduced fat or low calorie foods as well as fat replacers or substitutes have been the result of numerous attempts to meet the health recommendations without changing traditional ways of eating.
Three different types of fat replacers namely carbohydrate-based, protein-based and fat-based are reported in the literature.
Carbohydrate-based fat replacers consists of many products like dextrin, polydextrose, maltodextrin, cellulose, gums etc., which are used as thickeners and stabilizers in frozen desserts, salad dressings, margarine type spreads, baked products, frostings and snacks. Many products based on starch have been developed specifically as fat mimetic [Alexander, R. J., Cereal Food World 40 (1995) 366-368]. However, starch based products are not good for diabetics for whom good glucose control is necessary [Grundy, S. M., Diabetics Care 14 (1991) 796-801]. The U.S. Food and Drug Administration (FDA) regulations state that sensitive individuals may experience a laxative effect from excessive consumption of maltrodextrins [U.S.A. Food and Drug Administration Regulations 21 CFR Part 105]. Some popular examples of carbohydrate based fat replacers are Stellar, Remyrise AP, N-Oil, Lycadex, Maltrin, Ex-cel, Fibercel, Centu Tex, Fibrex etc. [Jones, J. M., Chemistry and Industry, (1996) 494-498].
Protein-based fat replacers are produced using common proteins such as egg white, skimmed milk or whey by microparticulating them into a particle size of 1-3 xcexcm to obtain a slippery and creamy fat like feeling which provide 1-2 kcal/g [Singer, N. S., et al., J.] Amer. Coll. Nutr. 9 (1990) 388-397]. These products are being used in variety of food products such as yogurts, cheese products, frozen desserts and also for formulating low fat baked goods such as cheese cakes and pie crust. Gelatin from fish waste was also reported as a fat replacer to use as a viscosity modifier and to impart a creamy texture. However, such fat replacers are reported to be hypersensitive for persons with allergy to the base proteins [Young, V. R., et al., J. Amer. Coll. Nutr. 9 (1990) 418-426]. Some examples of protein based fat replacers popular in the market are Dairylight, Simplesse, Lita, Calpra 75 etc. [Jones, J. M., Chemistry and Industry (1996) 494-498].
Carbohydrate-based and protein-based fat replacers are presently used in a range of foods, and are effective in delivering fat-like texture where the final product has a significant water content and is not exposed to extremely high temperatures or temperature variations [Mela, D. J., Fett/Lipid 98 (1996) 50-55]. These problems can be overcome by fat-based low calorie fats.
Fat-based low calorie fats have many advantages like functional and sensory properties very similar to the normal fats when compared to the carbohydrate and protein-based fat replacers. They also provide both the characteristic texture and flavour effects of native fats.
Many fat-based low calorie fats are reported in the literature namely propoxylated glycerols esterified with fatty acid chlorides [Masten, L. W., EP 571,219 (1993); White, J. F. et al., EP 325,010 (1989)]; fatty acid diesters of C4-10 dihydric alcohols [Klemann, L. P. et al., U.S. Pat. No. 5,286,512 (1994); Klemann, L. P. et al., U.S. Pat. No. 5,006,351 (1991)]; trioltriester derivatives [Klemann, L. P. et al., U.S. Pat. No. 5,043,179 (1991)]; polyol fatty acid polyesters [Kester, J. J. et al., U.S. Pat. No. 5,314,707 (1994); Letton, J. C., et al., 5,306,514 (1994)]; polyvinyloleate [D""Amelia, R. P. et al., U.S. Pat. No. 4,915,974 (1990)]; oleoylloeate [Jacklin, P. T. et al., U.S. Pat. No. 4,915,974 (1990)]; bis-oleoylaspartyladipare [Klemann, L. P. et al., U.S. Pat. No. 5,139,807 (1992)]; esterified alkoxylated mono- and diglycerides [Cooper, C. F. et al., U.S. Pat. No. 5,371,253 (1994)]; triglycerides containing C12-22 fatty acids having alkyl groups at least at the position 5,9,13 of the alkyl chain [Tagiri, M. et al., JP 04,325,055 (1992)]; 1,3-didecanoylglycerol [Mazur, A. W. et al., U.S. Pat. No. 5,137,660 (1992)]; alkyl or polyol thioesters [Klemann, L. P., U.S. Pat. No. 4,992,293 (1991)]; propyleneglycol diesters of medium chain and long chain saturated fatty acids [Stipp, G. K. et al., EP 495,553 (1992)]; alkylmalonic acid diesters [Fulcher, J. G. et al., Aus. Pat. No. 594,040 (1990)]; esterified polyoxyalkylene block co-polymers [Cooper, C. F. et al., EP 481,717 (1992)]; alkylglycoside fatty acid polyesters [Winter, D. D. et al., U.S. Pat. No. 4,942,054 (1990)]; fatty acid esters of sucrose [Letton, J. A., et al., EP 375,027 (1990)]; sorbitol fatty acid esters [Gruetzmacher, G. D., EP, 591,258 (1994)]; partially esterified polysaccharide with fatty acids [White, J. F. U.S. Pat. No. 4,959,466 (1990)]; alkoxylated sugar and sugar alcohol esters [Ennis, J. L. et al., EP 425,635 (1991)]; polysaccharide fatty acid polyester [Meyer, R. S. et al., U.S. Pat. No. 4,973,489 (1990)]. All these are unnatural compounds not normally encountered in human diet and the long term affects of consumption of such are presently unknown.
Examples of the more commonly known low calorie fats are OLESTRA(trademark), medium chain triglycerides (MCTs), Caprenin and SALATRIM(trademark). OLESTRA(trademark) is a mixture of hexa-, hepta-, and octa- fatty acid esters of sucrose. The physical properties of sucrose polyesters are similar to normal triglycerides [Jandacek, R. J., et al., Chem, Phys. Lipids 22 (1978) 163-176]. OLESTRA(trademark) is adaptable to most application where fats and oils are used. However, the major drawback to OLESTRA(trademark) is xe2x80x9canal leakagexe2x80x9d, the result of a non-digestible fat passing through the digestive system. OLESTRA(trademark) also blocks the absorption of fat soluble vitamins [Bailey""s Industrial Oil and fat Products, Vol. 1, Ed. Y. H. Hui (1996), p. 286; Jones D. Y. et al., Amer. J. Clin. Nutr. 53 (1991) 1282-1287 and Dasher, G., et al., FASEB J. 8 (1994) 443].
MCTs are triglycerols composed of C6, C8 and C10 saturated fatty acids. Hunder, J. E., et al., [U.S. Pat. No. 4,863,753 (1989)] reported a low calorie peanut butter containing xe2x89xa710% MCTs. A peanut butter composition containing MCTs (41.96%) was reported to have excellent consistency and contained at least 10% fewer calories than the normal fat. However, MCTs may be toxic and may induce metabolic acidosis in large doses [Akoh, C. C. Inform 6 (1995) 1055-1061].
SALATRIM(trademark) (short and long acylglycerol molecules) is a family of structured triacylglycerols prepared by interesterifying a completely hydrogenated vegetable oil with triacetin, tripropionin and/or tributyrin using sodium methoxide as a catalyst at 100-150xc2x0 C. [Wheeler, E. L. et al., U.S. Pat. No. 5,258,197 (1991); Klemann, L. P. et al., 42, J. Agr. Food Chem. (1994) 42, 442-446]. Thus, the SALATRIM(trademark) triglycerides are composed of mixtures of long-chain saturated fatty acids (predominantly stearic) and short-chain fatty acids (acetic propionic, and/or butyric) esterified to the glycerol backbone. A similar low calorie fat namely acetyl distearoyl glyceride was prepared by Wheeler, E. L. et al. [U.S. Pat. No. 5,258,197 (1991)]. Extensive testing in animals of SALATRIM(trademark) has shown no changes in the intestinal microflora or secondary bile acids, and no increased mutagenicity or other toxicological effects [Hayes, J. R., el al., J. Agr. Food Chem. 42 (1994) 500-514; Scheinbach, S., et al., ibid, 42 (1994) 572-580; Hayes, J. R., et al., ibid, 42 (1994) 539-551; Hayes, J. R., et al., ibid, 42 (1994) 515-520; and Hayes, J. R., et al., ibid, 42 (1994) 521-527]. However, SALATRIM(trademark) contains unnatural components such as very low molecular weight fatty acids and does not contain essential fatty acids.
Caprenin, a structured triglyceride consisting of caprylic and capric acids and the very long chain behenic acid which was developed for use in chocolate preparation. It yields only 5 cal/g instead of 9 cal/g because the short chain fatty acids have lower energy values and behenic acid is not well absorbed [Peters, J. C. et al., J. Am. Coll. Toxicol. 10 (1991) 357-67; Webb, D. R., et al., ibid 10 (1991) 341-356; Webb, D. R. et al, ibid., 10 (1991) 325-340]. Glycerin was esterified first with behenic acid to form glycerylmonobehenate, which was then reacted with capric and caprylic acids or their anhydrides, and then purified by molecular distillation and steam deodorization. Yoshida, T. et al., [JP 0559392 (1991)] reported the synthesis of 2-behenyl-1,3-dicaproyl glycerol by reacting tribehenin and ethyl caproate in the presence of lipase and the product""s feed study on rats showed that absorption of the structured triglycerides was significantly lower and its excretion into feces was higher than other oils. In another report Yoshida R., et al., [Shoka to Kyushu 14 (1991) 27-30 C. A. 117: 47249 (1992)] reported the synthesis of triglycerides with a randomly placed long chain fatty acid (behenic acid) and two medium chain fatty acids (capric and caproic acids) and included it in the diet of rats. These triglycerides were poorly absorbed from the intestine; absorption of behenic acid was particularly poor. Caprenin feeding studies have shown that it produces no toxic effects when fed as the primary source of dietary fat [Webb, D. R. et al., Food Chem. Toxicol. 31 (1993) 935-946]; Webb, D. R., et al., J. Amer. Coll. Toxicol. 10 (1991) 341-356 and Webb, D. R., et al, ibid, 10 (1991) 325-340]. However, a six week study of relative effects on serum lipids and apolipoproteins of a caprenin rich diet feeding studies by Wardlaw, G. M., et al., [Am. J. Clin. Nutr. 61 (1995) 535-542] showed that caprenin can contribute to hypercholesterolemia in men and gastrointestinal complaints in some individuals.
FIG. 1A is a graphical representation of mean body weight gain of all the test groups including the ad libitum group in the restricted diet growth experiment.
FIG. 2B is a graphical representation of the growth pattern of the 10% SO (control group) and 10% SL 3 (experimental group) in the ad libitum experiment.
The low calorie fats reported so far does not contain essential fatty acids and the natural antioxidants normally found in natural oils and fats. Hence, the objective of the present invention is to provide reduced-calorie fats which fulfils the three basic functions of fat in foods: (1) a source of essential fatty acids, (2) a carrier for fat soluble vitamins, and (3) a source of energy for storage or oxidation. This is achieved by incorporating poorly absorbable behenic acid into edible oils particularly vegetable oils such as sunflower, safflower and groundnut oils. A further objective of the present invention was to provide a reduced calorie plastic fat of the consistency of vanaspati and which does not contain the deleterious trans fatty acids.
In the present invention, reduced-calorie fats were prepared by incorporating behenic acid into edible oils using three different routes.
In the first two methods for the preparation of reduced-calorie plastic fats, 1,3-dibehenin prepared from mustard oil was used as a source of behenic acid. Other cruciferae oils such as rapeseed oil could also be used for this preparation. Mustard oil consists of about 44% of erucic acid along with other normal fatty acids. 1,3-Dierucin is prepared from mustard oil using the methodology developed by us previously [Kaimal T. N. B., et al. Biotechnology Letters 15, (1993) 353-356)] by lipase (from Candida cylindracea) hydrolysis under restricted water conditions and isolated 1,3-dierucin from the reaction mixture by crystallization from acetone at 10xc2x0 C. 1,3-Dierucin is hydrogenated to 1,3-dibehenin using conventional method with 10% palladium-carbon as catalyst under pressure (2-3 kg/cm2) in chloroform. After removal of the catalyst by filtration, 1,3-dibehenin is recrystallized from acetone.
The first route comprises interesterification of vegetable oils, exemplified by sunflower oil, and 1,3-dibehenin using sodium methoxide as catalyst at a concentration in the range of 0.3% to 0.5% by weight of the substrate at a temperature in the range of 80-150xc2x0 C. for a period in the range of 0.5 to 1.0 hour, purifying the product by silicic acid column chromatography to yield a reduced-calorie fat. The product thus prepared from sunflower oil and dibehenin in the molar ratio of 1:0.5 contained 29.8% of behenic acid and 44.6% of linoleic acid while a product containing 52.5% of behenic acid and 24.8% linoleic acid was obtained when the molar ratios were 1:1.
The second route provides a process for the preparation of reduced-calorie fat which comprises incorporation of sunflower oil fatty acids into the second position of 1,3-dibehenin to prepare a structured fat followed by its chemical interesterification with vegetable oils exemplified by sunflower oil. Accordingly this route consists of the following steps: a) saponification of sunflower oil to obtain fatty acid mixture, b) conversion of fatty acid mixture to anhydrides c) esterification of 1,3-dibehenin with the said fatty acid anhydrides to obtain structured fat, d) chemical interesterification of the structured fat with vegetable oils exemplified by sunflower oil, and e) isolation of reduced-calorie fat by silicic acid column chromatography.
Sunflower oil is saponified using conventional method by refluxing with 10% potassium hydroxide in ethanol and the saponified mass was neutralised with dilute hydrochloric acid and extracted with diethylether to obtain sunflower oil fatty acids. The sunflower oil fatty acid mixture was converted to their anhydride by treating with dicyclohexylcarbodiimide following conventional procedures. The fatty anhydride was then refluxed in chloroform with 1,3-dibehenin in presence of N,N-dimethylamino pyridine to obtain structured fat. The structured fat with a melting point of 55xc2x0 C. was then interesterified with vegetable oils exemplified with sunflower oil using sodium methoxide as catalyst at a concentration in the range of 0.3% to 0.5% by weight of the substrate and at a temperature in the range of 80-150xc2x0 C. for a period in the range of 0.5 to 1 hr till the randomization of the fatty acids was complete. The reduced-calorie fat was then purified by silicic acid column chromatography and found to contain about 32.1% of behenic acid and 33.7% of linoleic acid with a melting point of 36xc2x0 C.
The third route of the present invention provides a process for the preparation of reduced-calorie fat which comprises transesterification of vegetable oils and alkyl behenate with lipase [from Mucor miehei (Lypozyme 1M 20), 30 BIU/g, supplied by Novo Industri A/S, Denmark] and purifying the structured fat by silicic acid column chromatography to yield a reduced-calorie structured fat containing 5 to 41% of behenic acid and 33 to 60% of linoleic acid which is,an essential fatty acid. Only a slight excess of alkyl behenate (1.2 mole equivalents to that of vegetable oil) is used for transesterification. The interesterified reduced-calorie fat containing about 33% of behenic acid was found to have a slip melting point of about 37xc2x0 C. Various alkyl esters of behenic acid tried such as methyl, ethyl, isopropyl and n-butyl were used for this reaction but satisfactory levels of incorporation of behenic acid was obtained only when the ethyl esters were used.
The transesterification of sunflower oil and ethyl behenate is standardised by varying the enzyme concentration in the range of 1 to 4% by weight of the substrate, temperature in the range of 55-70xc2x0 C. and reaction period in the range of 1-6 hrs. Of the various parameters studied, enzyme concentration of 2%, temperature of 60xc2x0 C. and reaction period of 3 hr yielded a structured fat containing about 34% of behenic acid by GC analysis, and having a slip melting point of 37xc2x0 C.
Accordingly the present invention provides a process for the preparation of reduced-calorie fat which comprises esterification of edible oils with a source of behenic acid such as herein described, in presence of a catalyst at a temperature in the range 25xc2x0 C. to 150xc2x0 C. at least for 0.5 hr. and then recovering and purifying the reduced-calorie fats using conventional methods such as herein described. Edible oils which may be used are such as sunflower, groundnut, safflower, soybean, rapeseed, fish oil etc. The sources of behenic acid which may be used are 1,3-dibehenin, alkyl behenate and a structured fat containing behenic acid described herein. The esterification temperature may be in the range of 25xc2x0 C. to 150xc2x0 C. depending on the type of catalyst used which may be an alkali metal alkoxide or a thermostable lipase enzyme such as Lipozyme. The esterification reaction may be effected in the time range of 0.5 hr to 6 hr. Recovery of the reduced-calorie fat may be effected by filtration followed by column chromatographic purification in case of enzymatic transesterification or by washing-off the catalyst alkali metal alkoxide in case of chemical transesterification.
The following examples illustrate the invention and should not be construed as the limit of the invention and the manner in which it is carried out.